Live genetically attenuated malaria vaccine

ABSTRACT

Method for inoculating a vertebrate host against malaria, by administering to the host a live  Plasmodium  organism that is genetically engineered to disrupt a liver-stage-specific gene function.

This application claims the benefit of U.S. Provisional Application No.60/631,228, filed Nov. 26, 2004, and U.S. Provisional Application No.60/531,479, filed Dec. 19, 2003, both of which are incorporated byreference in their entireties.

STATEMENT OF GOVERNMENT LICENSE RIGHTS

This invention was made with government support under A1053709 awardedby the National Institutes of Health. The government has certain rightsin the invention.

FIELD OF THE INVENTION

This invention relates to live genetically modified Plasmodium organismsand their use as immunospecific immunoeffectors for vaccinationpurposes.

BACKGROUND OF THE INVENTION

Malaria has a tremendous impact on human health, killing millionsannually and the disease is a major impediment for social and economicdevelopment of nations in malaria-endemic areas, particularly insub-Saharan Africa (1, see the appended Citations). Malaria is amosquito-borne disease that is transmitted by inoculation of thePlasmodium parasite sporozoite stage. Sporozoites invade hepatocytes(2), transform into liver stages, and subsequent liver stage developmentultimately results in release of pathogenic merozoites (3).

Because an effective ‘subunit’ malaria vaccine has remained elusive andthe complexity of the malaria parasite Plasmodium might preclude thesuccessful development of such a vaccine, whole organism vaccineapproaches against malaria have lately found renewed interest (4). Thefeasibility of such a vaccine has been demonstrated in animal models andsubsequently in humans by induction of sterile protective immunitythrough inoculation with irradiation-attenuated parasites (5, 6). Liverstages are a prime malaria vaccine target because they can be completelyeliminated by sterilizing immune responses, thereby preventing malariainfection (7). The recent availability of complete Plasmodium genomesequences (8, 9) may now permit the development of live-attenuatedparasites by more precise and defined genetic manipulations.

Using expression profiling, we previously identified genes that arespecifically expressed during the pre-erythrocytic part of the parasitelife cycle (11, 12). A number of pre-erythrocytic genes named UIS(up-regulated in infective sporozoites) also showed up- regulation insporozoites when they gain infectivity for the mammalian host (11).

SUMMARY OF THE INVENTION

Here we show by reverse genetics that selected individual genes,exemplified by UIS3 (up-regulated in infective sporozoites gene 3) andUIS4, are essential for early liver stage development: uis3(−) anduis4(−) sporozoites infect hepatocytes but are no longer able toestablish blood stage infections in vivo and thus do not lead todisease. The invention thereby provides the first live Plasmodiumorganisms that are genetically engineered to disruptliver-stage-specific gene functions

Surprisingly, immunization with either uis3(−) or uis4(−) sporozoitesconfers complete protection against infectious sporozoite challenge in arodent malaria model. This protection is sustained and stage-specific.These findings provide the first genetically attenuated whole organismmalaria vaccines.

Thus, the invention provides a method for inoculating a vertebrate hostagainst malaria, by administering to the host a live Plasmodium organismthat is genetically engineered to disrupt a liver-stage-specific genefunction. The invention further provides a vaccine compositioncomprising a live Plasmodium organism that is genetically engineered todisrupt a liver-stage-specific gene function. In addition, the inventionprovides the use of a vaccine composition comprising a live Plasmodiumorganism that is genetically engineered to disrupt aliver-stage-specific gene function. The invention also provides forproduction of a vaccine composition, by suspending the subjectengineered Plasmodium organisms in a suitable pharmaceuticallyacceptable carrier solution.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of thisinvention will become more readily appreciated as the same become betterunderstood by reference to the following detailed description, whentaken in conjunction with the accompanying drawings, wherein:

FIG. 1 depicts the primary structure of Plasmodium UIS3 proteins, asdescribed in Example 1; and

FIG. 2 depicts the replacement strategy used to generate the uis3(−)parasite described in Example 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The invention provides a method for inoculating a vertebrate hostagainst a Plasmodium parasite, by administering to the host a livePlasmodium organism that is genetically engineered to disrupt aliver-stage-specific gene function.

By Plasmodium parasite is meant any member of the protozoan genusPlasmodium, including the four species that cause human malaria: P.vivax, P. malariae, P. falciparum, and P. ovale. The correspondingvertebrate host is a human or other secondary host that is susceptibleto infection by the wild-type Plasmodium parasite.

For use as a live anti-malarial vaccine, the Plasmodium parasite isgenetically engineered to disrupt a liver-stage-specific gene function.The term “disrupt liver-stage-specific gene function” or “disruptLS-specific gene function” means interfering with an LS-specific genefunction such as to completely or partially inhibit, inactivate,attenuate, or block the LS-specific gene function, for example, by genedisruption or influencing transcription, translation, protein folding,and/or protein activity. The term “liver-stage-specific gene function”or “LS-specific gene function” refers to a function that is required inliver stage parasites to ultimately produce infectious merozoites andestablish the erythrocytic stage of the life cycle, but that is notrequired for entry into host hepatocytes or maintenance of the parasitein asexual blood cell stages and production of infective sporozoites.Malaria infection is initiated by Plasmodium sporozoites in the salivaryglands of mosquitoes. These sporozoites invade hepatocytes of thevertebrate host and differentiate into liver stage (LS) forms. After afew days the LS parasites produce several thousand merozoites that arereleased from the hepatocytes and invade erythrocytes to start the bloodstage cycle that causes malaria disease. According to the invention, thePlasmodium parasite is genetically engineered to disrupt at least oneLS-specific gene function such that the genetically engineered parasitesremain capable of invading hepatocytes but cannot produce merozoitesthat can establish blood stage infections.

An LS-specific gene function may be identified using routine methodologythat is standard in the art. For example, an LS-specific gene functionmay be identified by assessing the function of genes whose expression isup-regulated in liver-stage parasites (LS-up-regulated genes). Forexample, genes whose expression is up-regulated in liver-stage parasitesmay be expressed at higher levels in liver-stage parasites than in thesporozoite population that emerges from mosquito mid-gut oocysts.Up-regulation of expression of such genes may also be observed inmature, infective salivary gland sporozoites (like in the UIS4 and UIS3genes discussed in the Examples below). Well-known methods fordifferential transcriptional profiling, including, but not limited to,subtractive hybridization screens, differential display, and genome-widemicroarray analyses, may be used for identifying genes whose expressionis up-regulated in liver-stage parasites. Such methods have beenpreviously used to analyze infectivity-associated changes in thetranscriptional repertoire of sporozoite-stage parasites (11) and toidentify Plasmodium genes that encode pre-erythrocytic stage-specificproteins (12). For example, suppression subtractive hybridizationpermits selective enrichment of differentially regulated cDNAs of highand low abundance through a combination of hybridization and polymerasechain reaction (PCR) amplification protocols that allow the simultaneousnormalization and subtraction of the cDNA populations. Suppressionsubtractive hybridization has been used to analyze transcriptionaldifferences between non-infective and infective sporozoites and toidentity genes controlling infectivity to the mammalian host (11). Thisprocedure has permitted the identification of LS-up-regulated genes,including, but not limited to, UIS3 and UIS4, as further described inthe Examples below. Suppression subtractive hybridization of Plasmodiumsalivary gland sporozoites versus merozoites has also been used toidentify stage-specific pre-erythrocytic transcripts (12). Differentialexpression of candidate LS-specific genes may be confirmed using methodsthat are standard in the art, including dot blots, reverse transcriptasePCR (RT-PCR), immunoblotting, immunofluorescence microscopy, and/ormicroarray expression analyses, as previously described (11, 12).

In some embodiments of the invention, LS-specific gene functions areidentified by analyzing the function of LS-up-regulated genes, asfurther described below. However, not all genes with an LS-specific genefunction are necessarily LS-up-regulated genes. Thus, genes whoseexpression is not up-regulated in LS forms may nevertheless possess anLS-specific gene function.

Interference with a liver-specific function may also be achieved byLS-specific overexpression of an inhibitory factor. This factor may beinserted by reverse genetics methods into a pseudogene, i.e., one thatis not essential for parasite survival at any time point during the lifecycle (47). The inhibitory factor should not confer toxicity to theparasite but rather act in arresting LS development. Such a factor mayinclude, but is not limited to, inhibitors of cell-cycle progressionand/or ubiquitin-mediated proteolysis, and/or factors that interferewith post-transcriptional control of gene-expression.

LS-specific gene functions may be identified by analyzing the phenotypeof parasites in which one or more gene functions have been disrupted.Several methods for disrupting gene functions in Plasmodium arewell-known in the art and may be used in the practice of the invention.Such methods include, but are not limited to, gene replacement byhomologous recombination, antisense technologies, and RNA interference.For example, methods of gene targeting for inactivation or modificationof a Plasmodium gene by homologous recombination have been previouslydescribed (13). Such methods are herein successfully used to disruptLS-specific gene functions, as described in Examples 1 and 2. Antisensetechnology has also been successfully used for disrupting Plasmodiumgene functions. For example, exogenous delivery of phosphorothioateantisense oligonucleotides against different regions of the P.falciparum topoisomerase II gene result in sequence-specific inhibitionof parasite growth (14). Similarly, transfection of an antisenseconstruct to the Plasmodium falciparum clag9 gene, which had been shownto be essential for cytoadherence by targeted gene disruption, resultedin a 15-fold reduction in cytoadherence compared to untransfectedcontrol parasites (15).

Another exemplary technology that may be used in the practice of theinvention to disrupt LS-specific gene functions is RNA interference(RNAi) using short interfering RNA molecules (siRNA) to producephenotypic mutations in genes. RNAi has been used as a method toinvestigate and/or validate gene function in various organisms,including plants, Drosophila, mosquitoes, mice, and Plasmodium (see,e.g., 37-44) In Plasmodium, RNAi has been used, for example, todemonstrate the essential role of a PPI serine/threonine proteinphosphatase (PfPP1) from P. falciparum (41). RNAi has also been used toinhibit P. falciparum growth by decreasing the level of expression ofthe gene encoding dihydroorotate dehydrogenase (42) and by blocking theexpression of cysteine protease genes (43). In the mouse malaria model,RNAi has been used to inhibit gene expression in circulating P. bergheiparasites in vivo (44). These studies have demonstrated the use of RNAias an effective tool for disrupting gene function in Plasmodiumorganisms.

The gene disruption approaches described above (for example, genetargeting by homologous recombination, antisense, and RNAi) have beenused successfully to investigate the function of virtually all genes inan organism's genome. For example, the availability of sequenced genomeshas enabled the generation of siRNA libraries for use in large-scaleRNAi studies to screen for genes that are involved in various processes,such as developmental pathways or stages (see, e.g., 45 and 46). Suchscreens may be used in the practice of the invention to identifyLS-specific gene functions in Plasmodium. Assays that may be used foridentifying LS-specific gene functions include, but are not limited to,phenotypic analyses such as the phenotypic assays described in Examples1 and 2. The term ‘phenotypic analysis’ includes all assays with vitalrecombinant parasites that are generated in a wild type, fluorescent orany other transgenic reporter background. Assays may be performed invivo, with cultured cells, in in vitro development assays or any othersystem that provides a read-out for LS development.

The engineered Plasmodium organisms in which an LS-specific genefunction has been disrupted are typically grown in cell culture oranimals, expanded in the mosquito host, and harvested as sporozoites foruse in vaccines (see, e.g., 16).

The subject vaccine compositions are produced by suspending theattenuated live Plasmodium organisms in a pharmaceutically acceptablecarrier. Suitable pharmaceutically acceptable carriers include sterilewater or sterile physiological salt solution, particularly phosphatebuffered saline (PBS), as well known in the art.

Vaccines according to the invention can be administered, e.g.,intradermally, subcutaneously, intramuscularly, intraperitoneally, andintravenously.

Dosage is empirically selected to achieve the desired immune response inthe host. By immune response is meant an acquired and enhanced degree ofprotective immunity, preferably complete or sterile protection, againstsubsequent exposure to wild-type Plasmodium sporozoites. In the workingexamples described below, sterile protection was achieved followingthree vaccinations with 10,000 live genetically attenuated sporozoitesper inoculation.

DETAILED TECHNICAL DESCRIPTION

Background. Radiation-attenuated sporozoites are a singular model thatachieves sterile, protective immunity against malaria infection.

Malaria causes more than 300 million clinical cases and more than 1million death annually. The disease has a severe negative impact on thesocial and economic progress of developing nations. Transmission of themalaria parasite Plasmodium to the mammalian host occurs when infectedmosquitoes bloodfeed and inoculate the sporozoite stage (spz). Afterentering the bloodstream, spzs are quickly transported to the liverwhere they extravasate and invade hepatocytes (2). Within hepatocytes,spzs transform into liver stages (LS) (also called exo-erythrocyticforms, EEFs). LS parasites grow, undergo multiple rounds of nucleardivision and finally produce thousands of merozoite (17, 18). Merozoitesreleased from the liver rapidly invade red blood cells and initiate theerythrocytic cycle, which causes malaria disease. A protective malariavaccine would have tremendous impact on global health but despite over acentury of efforts, no vaccine has been developed that confers prolongedprotection. Yet, we have known for more than 35 years that sterileprotracted protection against malaria infection is possible.

Immunization of mice with radiation-attenuated rodent model malaria spzs(gamma-spzs) induces sterile immunity against subsequent infectious spzchallenge, thus completely preventing the initiation of blood stageinfection from the liver (5). Importantly, based on these findings itwas later shown that immunization of humans with gamma-P. falciparumspzs completely protected greater than 93% of human recipients (13 of14) against infectious spz challenge and that protection can last for atleast 10 months (6). Gamma-spzs retain the capacity to infect the liverof the mammalian host and invade hepatocytes (19-20). However, LSderived from gamma-spzs suffer arrested development and thus do notproduce red blood cell-infectious merozoites. Although, the inoculatedstage is the spz, the main immune target is the infected hepatocyteharboring the LS (21). Protective immunity is spz dose and radiationdose dependent: greater than 1000 immunizing bites from P.falciparum-infected mosquitoes exposed to 15,000-20,000 rads of gammaradiation is required to protect the majority of subjects exposed toinfectious spz challenge (6). Mosquitoes inoculate between 10-100 spzsduring a bite (22-23). Therefore, the total spz dose for completeprotection comes to 10,000-100,000. Importantly, immunization withover-irradiated spzs or heat-inactivated spzs fails to induceprotection, indicating that the spz must remain viable for some timeafter inoculation and must progress to a liver stage that inducesprotection (6, 24). On the basis of observations in the rodent malariamodel, protracted protective immunity may depend on sufficientexpression of LS antigen (Ag), because treatment with primaquine, a drugthat kills LS, aborts the development of protection (21). Importantly,protection induced by P. falciparum gamma-spzs is strain-transcending:inoculation with gamma-spzs of one parasite strain confers protectionagainst heterologous strains (6).

Although we have learned much about spz gene expression in the last fewyears (25-27) the LS as the true immunological target of gamma-spzsinduced protection have so far completely eluded gene expressionanalysis because of their inherent experimental inaccessibility. Wecurrently know only one liver stage-specific Ag, liver stage antigen-1(LSA-1) (28). Thus, the fine Ag specificity of lymphocytes participatingin protective immunity remains unknown in humans, because the Agsexpressed by LS parasites remain unknown.

Feasibility to create genetically attenuated Plasmodium Liver Stages. Togenerate genetically attenuated Plasmodium LS that are defective only inLS development a stage-specific gene that plays an essential andexclusive role at this stage needs to be disrupted. The gene cannot beessential during the blood stage cycle given that Plasmodium is haploidand transfection is done with asexual blood stages and the mutantparasites are maintained as blood stages (13). We previously employedtranscription-profiling based on the prediction that infectiousPlasmodium spzs residing in the mosquito salivary glands are uniquelyequipped with transcripts required for hepatocyte invasion andsubsequent development of the LS (11). Next, we screened for transcriptsthat are specific for pre-erythrocytic and absent from blood cell stagesin order to generate a subset of genes that can disrupted (12). Thecombined screens identified two abundant salivary gland spz enrichedtranscripts that are absent from blood stages, termed UIS3 and UIS4 (forupregulated in infectious spzs). Cell biological studies showed thatboth encoded proteins locate to the parasitophorous vacuole, theparasite-derived organelle where replication and schizogony takes place(data not shown).

Gene knockouts using insertion and replacement strategies have nowrevealed that both genes are necessary for LS development (see Examples1 and 2 below). Both proteins are already expressed in spzs (data notshown) but uis3(−) and uis4(−) parasites develop normal spzs and theseinvade hepatocyte normally. However, uis3(−) and uis4(−) LS arrest inintermediate-LS development and do not produce late LS (data not shown).Therefore, both UIS3 and UIS4 have LS-specific gene functions.Importantly, animals infected by natural bite or intravenously withdoses of up to 10,000 spzs do not become patent, confirming that bothgenes play vital roles in successful completion of the Plasmodium lifecycle (see Tables 1 and 2 below). Therefore, we succeeded in generatingthe first genetically attenuated LS. Based on these discoveries we andothers can now advance and test various LS-up-regulated genes identifiedby microarray analysis for their importance in LS development. Wepredict that more LS-up-regulated genes will turn out to be essentialfor LS development (i.e., to possess LS-specific gene functions),especially uniquely expressed genes given the remarkable capacity of theparasite to develop from a single spz to more than 10,000 daughtermerozoites. Such LS-up-regulated genes can be similarly disrupted toproduce additional live vaccine candidates, as described herein.

Representative embodiments of the present invention are described in thefollowing two working examples.

EXAMPLE 1

This first Example was published by Nature AOP on Dec. 5, 2004 (29).

We hypothesized that inactivation of UIS genes for which expression isrestricted to pre-erythrocytic stages could lead to attenuation of theliver stage parasite, without affecting the blood stages or mosquitostages. We focused on a gene called UIS3 that encodes a small conservedtransmembrane protein (FIG. 1). UIS3 was expressed in infectioussporozoites (12) and we determined that it was also expressed aftersporozoite infection of livers in vivo (data not shown). UIS3 of rodentmalaria parasites (accession number EAA22537) and UIS3 of the humanmalaria parasite P. falciparum (Pf13_(—)0012) show 34% amino acidsequence identity (FIG. 1). Because the rodent malaria parasites such asP. berghei (Pb) are excellent models to study Plasmodium liver stage andpre-erythrocytic immunity we pursued investigation of UIS3 in thisspecies.

The endogenous PbUIS3 gene was deleted using a replacement strategy (13)(FIG. 2). After transfection, parental blood stage parasites were usedto obtain clonal parasite lines designated uis3(−) that containedexclusively the predicted locus deletion (data not shown). As expected,uis3(−) parasites showed normal asexual blood stage growth and normaltransmission to the Anopheles mosquito vector (data not shown). Withinthe mosquito uis3(−) sporozoites developed normally in midgut oocyctsand infected the salivary glands in numbers comparable to wildtype (WT)sporozoites (data not shown). Reverse transcriptase (RT)-PCR confirmedlack of UIS3 expression in uis3(−) sporozoites (data not shown). uis3(−)sporozoites showed typical gliding motility, a form ofsubstrate-dependant locomotion that is critical for sporozoitetransmission and infectivity (30) (data not shown). They also retainedtheir host cell invasion capacity of cultured hepatoma cells at levelscomparable to WT parasites (data not shown).

Intracellular uis3(−) sporozoites initiated the typical cellulartransformation process that leads to de-differentiation of thebanana-shaped elongated sporozoite to a spherical liver trophozoite (17,31) (data not shown). In marked contrast, uis3(−) parasites showed asevere defect in their ability to complete transformation into livertrophozoites (data not shown). Only a small fraction of uis3(−)parasites developed into spherical early liver stages that also appearedconsistently smaller than the corresponding WT forms. Consequently,mutant parasites lacked the capacity to progress to mature liverschizonts (data not shown). Based on this extreme developmental defectobserved in vitro, we next tested if uis3(−) sporozoites had lost theircapacity to progress through liver stage development and lead to bloodstage infections in vivo. Indeed, intravenous injection of up to 100,000uis3(−) sporozoites failed to induce blood stage parasitemia in youngSprague/Dawley rats which are highly susceptible to P. bergheisporozoite infections (data not shown). Control WT sporozoites inducedblood stage parasitemia in rats between 3-4 days after injection.

Thus, the observed phenotypic characteristics of uis3(−) parasites,i.e., their ability to invade hepatocytes and their defect in completeliver stage development allowed us to test them as a whole organismvaccine in a mouse/sporozoite challenge model. We intravenouslyimmunized mice with uis3(−) sporozoites using different prime-boostregimens and subsequently challenged the mice by intravenous injectionof infectious WT sporozoites (Table 1). Protection was evaluated byblood smear to detect the development of blood stage parasitemiastarting two days after sporozoite challenge, the most stringent readoutfor sterile protection against malaria infection. Priming with 50,000uis3(−) sporozoites followed by 2 boosts with 25,000 uis3(−) sporozoitescompletely protected all immunized mice against a challenge with 10,000WT sporozoites given 7 days after the last boost (Table 1). Completesterile protection against the same sporozoite challenge dose was alsoachieved with a similar prime-2 boost protocol using 10,000 uis3(−)sporozoites (Table 1). We next immunized mice using the same prime-boostprotocols but challenged with WT sporozoites 4 weeks after the lastboost. None of the challenged mice developed blood stage infections andthus enjoyed protracted sterile protection (Table 1). Protractedprotection was confirmed by a re-challenge experiment where protectedanimals were challenged again with a high inoculum of 50,000 infectioussporozoites after two months. All animals remained completely protected.Mice immunized with uis3(−) sporozoites were also completely protectedagainst re-challenge by infectious mosquito bite (Table 1). To determinethe level of protection with a reduced immunization dose we tested aprime-single boost protocol with 10,000 uis3(−) sporozoites. Seven outof ten animals enjoyed complete protection, while the remaining threeanimals became patent after a long delay in patency. Next, a subset ofimmunized mice was challenged by direct inoculation with blood stageparasites. All animals developed blood stage parasitemia two days afterchallenge, indicating that the observed protective immunity is notacting against blood stages and thus was specific againstpre-erythrocytic stages. Finally, to evaluate a more vaccine-relevantdelivery route we immunized mice subcutaneously using a prime-2 boostprotocol with 50,000 uis3(−) and 25,000 uis3(−) sporozoites,respectively. All mice were completely protected against subsequentintravenous WT sporozoite challenge.

Our results show that it is possible to develop genetically modifiedmalaria parasites that are completely attenuated at the liver stage,which normally establishes infection of the mammalian host aftermosquito transmission. This attenuation was achieved by deletion of asingle parasite gene, UIS3. Although UIS3 function remains unknown,uis3(−) parasites clearly lacked the ability to compensate for its loss.The protracted sterile protection against malaria that we observed afterimmunization with uis3(−) sporozoites in the mouse/sporozoite challengemodel provides proof of principle that a genetically modified malariavaccine is feasible. We identified a UIS3 orthologue (accession numberPF13_(—)0012) in the genome of the most lethal human malaria parasite P.falciparum. This will allow us to create a genetically attenuateduis3(−) human parasite that can be tested as a vaccine inhuman/sporozoite challenge models. Together our findings lead the way tothe development of a genetically attenuated, protective whole organismmalaria vaccine that prevents natural infection by mosquito bite.

Methods: Plasmodium berghei transfection. For replacement of PbUIS3 twofragments were amplified using primers: UIS3rep1for (5′GGGTACCCGCATTAGCATAACATCTCATTGG 3′) (SEQ ID NO: 1) and UIS3rep2rev (5′CAAGCTTGCTTTCATATATTTGTTATTTGTC 3′) (SEQ ID NO: 2) for the 800 bp 3′fragment; and: UIS3rep3for (5′ GGAATTCCCATATGTTTGTGTAACATC 3′) (SEQ IDNO: 3) and UIS3rep4rev (5′ CTCTAGAGTGTGCTTAAATGTTTCTTTAAAC 3′) (SEQ IDNO: 4) for the 760 bp 5′ fragment using P. berghei genomic DNA astemplate. Cloning into the P. berghei targeting vector (13) resulted inplasmid pAKM19. To obtain clonal parasite populations, limited dilutionseries and i.v. injection of one parasite into 15 recipient NMRI miceeach was performed. For RT-PCR analysis we dissected 6×10⁵ uis3(−) and6×10⁵ WT salivary gland sporozoites and isolated polyA⁺ RNA using oligodT-columns (Invitrogen). For cDNA-synthesis and amplification weperformed a two step-PCR using random decamer primers (Ambion) andsubsequent standard PCR reactions.

Phenotypical analysis of uis3(−) parasites. Anopheles stephensi mosquitorearing and maintenance were under a 14 h light/10 h dark cycle, 75%humidity and at 28° C. or 20° C., respectively. For each experiment,mosquitoes were allowed to blood-feed for 15 min. on anaesthetizedNMRI-mice that had been infected with wild-type P. berghei NK65 or theuis3(−) clone and were assayed for a high proportion of differentiatedgametocytes and microgametocyte-stage parasites capable ofexflagellation. Mosquitoes were dissected at days 10, 14, and 17 todetermine infectivity, midgut sporozoite and salivary gland sporozoitenumbers, respectively. For analysis of sporozoite motility, sporozoiteswere deposited onto precoated (3% BSA/RPMI 1640) glass coverslips, fixedfor 10 min at RT with 4% paraformaldehyde, and incubated using primaryantibody against P. berghei circumsporozoite protein (anti-PbCSP) (32).To detect liver stages in hepatocytes, 10³ Huh7 cells were seeded ineight chamber slides and grown to semiconfluency. P. berghei sporozoiteswere added, incubated 90 min. at 37° C., and washed off. After 8, 12,15, 24, 36 and 48 h, LS were revealed using primary antibodies againstthe P. berghei heat shock protein 70 (HSP70) (33). To analyze sporozoiteinvasion a double staining protocol with anti-CSP antibody was used(34). To determine the infectivity of clonal sporozoite populations invivo young Sprague-Dawley rats were injected intravenously with 100microliter sporozoite suspension in RPMI 1640. Parasitemia of theanimals was checked daily by Giemsa-stained blood smears. The appearanceof a single erythrocytic stage represents the first day of patency.

Immunization and parasite challenge experiments. For all experimentsfemale C57BL/6 mice (Charles River Laboratories) at the age of 50 to 80days were used. For immunization, uis3(−) sporozoites were extractedfrom salivary glands from infected mosquitoes. Typically, a singleinfected mosquito contained 20,000 uis3(−) sporozoites. Sporozoites wereinjected in a volume of 100 microliters intravenously into the tail veinor subcutanously into the neck of animals. Animals were immunized with asingle dose of 1 or 5×10⁴ uis3(−) sporozoites, followed by two boosts ofeither 1 or 2.5×10⁴ uis3(−) sporozoites administered i.v. or s.c. Thefirst boost was given 14 days following the immunization, with a secondboost following 7 days thereafter, or at time intervals indicated. Oneset of animals was immunized followed by a single boost with 1×10⁴uis3(−) sporozoites each. The animals were then monitored for theparasitemia by daily blood smears. All animals remained blood stageparasite-negative after the first immunization and subsequent boosts.Animals were challenged 7 days up to 1 month after receiving the lastboost of uis3(−) sporozoites by intravenous or subcutanous injection ofeither 5×10⁴ or 1×10⁴ infectious P. berghei WT sporozoites. For each setof experiments, at least three naive animals of the same age group wereincluded to verify infectivity of the sporozoite challenge dose. In eachnaive animal, parasitemia was readily detectable at days three to fiveafter injection by Giemsa-stained blood smears. Protected animals weremonitored for at least 14 days and typically up to 1 month. Are-challenge study was performed for one immunization experiment twomonths after the first challenge with a single dose of 5×10⁴ infectiveP. berghei WT sporozoites. To test whether uis3(−) immunized mice wereprotected against re-challenge by natural transmission 10 protected and5 naive control mice were exposed for 10 min to 10 highly infectedmosquitoes that contained an average of 40,000 WT salivary glandsporozoites each. Successful blood-feeding was confirmed by mosquitodissection after the challenge experiment. To confirm stage-specificityof protection, an additional experiment was performed with 10 mice thatwere fully protected against a challenge with infective sporozoites. Allimmunized mice and three naive control mice were challenged byintravenous injection of 5×10⁴ P. berghei WT blood stage parasites. Allmice were fully susceptible to blood stage inoculations with nodifferences in patency.

Results: Table 1 below shows that C57B1/6 mice immunized with P. bergheiuis3(−) sporozoites are completely protected against a challenge with WTP. berghei sporozoites.

TABLE 1 # Protected/ Immunization Boosts: 1st/2nd Challenge dose #Challenged Exp. #'s uis3(—) spz. numbers (day) (timepoint) (pre-patency)I. 50,000 25,000 (d.14)/25,000 10,000 spz. (d.7) 10/10 (d.21) (noinfection) 10,000 10,000 (d.14)/10,000 10,000 spz. (d.7) 10/10 (d.21)(no infection) — — 10,000 spz. 0/9 (d.3) II. 50,000 25,000 (d.34)/25,00010,000 spz. (d.30) 5/5 (no infection) (d.45) 10,000 10,000 (d.34)/10,00010,000 spz. (d.30) 5/5 (no infection) (d.45) — — 10,000 spz. 0/6 (d.4.5)IIII. 50,000 50,000 (d.14)/10,000 10 inf. mosq. (d.38) 5/5 (noinfection) (d.21) 10,000 10,000 (d.14)/10,000 10 inf. mosq. (d.38) 5/5(no infection) (d.21) — — 10 inf. mosq. 0/5 (d.3) IV 10,000 10,000(d.14)/— 10,000 spz. (d.7) 7/10 (d.8) — — 10,000 spz. 0/5 (d.3) V.50,000 25,000 (d.14)/25,000 10,000 blood st. 0/5 (d.2) (d.21) (d.30)10,000 10,000 (d.14)/10,000 10,000 blood st. 0/5 (d.2) (d.21) (d.30) — —10,000 blood st. 0/3 (d.2) VVI. 50,000 s.c. 25,000 (d.11) s.c./ 10,000spz. (d.23) 5/5 (no infection) 25,000 (d.18) s.c. 50,000 s.c. 25,000(d.11) s.c./ 50,000 spz. (d.23) 5/5 (no infection) 25,000 (d.18) s.c. —10,000 spz. 0/6 (d.4.5) Notes: Mice were immunized with P. bergheiuis3(—) sporozoites. Mice were challenged with infectious P. berghei WTsporozoites or blood stages. Mice were from the same age group (50-80days old) and sporozoites were from the same mosquito batch. Timepointsin column 4 indicate the day of challenge after the final boost. Thepre-patent period is defined as the time until the first appearance of asingle erythrocytic stage in Giemsa-stained blood smears. Five mice ofthe Exp. I. group were re-challenged with one dose of 50,000 WTsporozoites 2 months after the first challenge and remained protected.

EXAMPLE 2

Here, we disrupt another Plasmodium protein with a critical function forcomplete liver stage development. UIS4 (upregulated in infectivesporozoites gene 4) is expressed exclusively in infective sporozoitesand developing liver stages. Targeted gene disruption of UIS4 in therodent model malaria parasite Plasmodium berghei generated knockoutparasites that complete the malaria life cycle until after hepatocyteinvasion. UIS4 knockout parasites transform into early liver stages.However, they are severely impaired in further liver stage developmentand can only initiate blood stage infections when inoculated at highsporozoite doses. Immunization with UIS4 knockout sporozoites completelyprotects mice against subsequent infectious wildtype sporozoitechallenge. After sporozoite invasion of hepatocytes, UIS4 localizes tothe newly formed parasitophorous vacuole membrane that constitutes theparasite-host cell interface and extends as a tubo-vesicular networkinto the hepatocyte cytoplasm. Together our data demonstrate thatdepletion of UIS4 results in attenuated liver stage parasites.Genetically attenuated liver stages may induce immune responses, whichinhibit subsequent infection of the liver with wildtype parasites.

Generation of uis4(−) parasites. Given that UIS4 is expressed insporozoites but not in blood stages, we were able to pursue a targetedgene disruption at the blood stages to study the importance of UIS4 forthe Plasmodium pre-erythrocytic life cycle stages. The endogenous PbUIS4gene was disrupted using the above-described insertion and replacementstrategies (data not shown). The parental blood stage population fromthe successful transfection was used for selection of clonal parasitelines carrying the gene disruption. We obtained insertion/disruptionclones designated uis4(−) and replacement clones designated uis4REP(−)that contained exclusively the predicted mutant locus. The correctreplacement event was confirmed by insertion-specific PCR (data notshown). To confirm PbUIS4 deficiency of the mutant parasites weperformed RT-PCR and cDNA amplification using polyA⁺RNA from salivarygland sporozoites as templates (data not shown). Moreover, Western blotanalysis of uis4REP(−) sporozoites did not detect PbUIS4 (data notshown).

Plasmodium berghei transfection and genotypic analysis. For genetargeting of PbUIS4 a 582 bp fragment was amplified using primersUIS4INT for (5′ CGGAATTCATCATATTACTAATTTTCGGGGG 3′) (SEQ ID NO: 5) andUIS4INTrev (5′ TCCCCGCGGTTATTCCATGTTATAAACGTTATTTCC 3′) (SEQ ID NO: 6)using P. berghei genomic DNA as template. Cloning into the P. bergheitargeting vector (13) resulted in plasmid pAKM15. Parasitetransformation and selection was performed as described previously (13).Integration-specific PCR amplification of the uis4(−) locus was achievedusing the following primers: test 1, T. gondii DHFR-TS for (5′CCCGCACGGACGAATCCAGATGG 3′) (SEQ ID NO: 7) and UIS4 test rev (5′CCCAAGCTTAGTTTGCATATACGGCTGCTTCC 3′) (SEQ ID NO: 8); test 2, UIS4 testfor (5′ CGGAATTCTGGATTCATTTTTTGATGCATGC 3′ (SEQ ID NO: 9) and T7 (5′GTAATACGACTCACTATAGGC 3′) (SEQ ID NO: 10). For replacement of PbUIS4 twofragments 1 kb and 600 bp were amplified using primers UIS4rep1 for (5′GAATTCTGGATTCATTTTTTGATGCATGC 3′) (SEQ ID NO: 11) and UIS4rep2rev (5′GGGGTACCTTTATTCAGACGTAATAATTATGTGC 3′) (SEQ ID NO: 12) for the 1 kbfragment and UIS4rep3 for (5′ AAAACTGCAGATAATTCATTATGAGTAGTGTAATTCAG 3′)(SEQ ID NO:13) and UIS4rep4rev (5′ CCCCAAGCTTAAGTTTGCATATACGGCTGCTTCC3′) (SEQ ID NO:14) for the 600 bp fragment using P. berghei genomic DNAas template. Cloning into the hDHFR targeting vector (34) resulted inplasmid pAKM17. To detect UIS4 expression in WT and mutant P. bergheiparasites, 1×10⁵ salivary gland sporozoites were dissolved in 10microliters SDS sample buffer. UIS4 was visualized on Western blotsusing the polyclonal UIS4 antisera (12) and horseradishperoxidase-coupled anti-rabbit IgG secondary antibody (Amersham). ForRT-PCR analysis we dissected 8×10⁵ uis4(−), 8×10⁵ uis4REP(−) and 4×10⁵WT salivary gland sporozoites and isolated polyA⁺ RNA using oligodT-columns (Invitrogen). For cDNA synthesis and amplification weperformed a two step-PCR using random decamer primers (Ambion) andsubsequent standard PCR reactions.

Phenotypic analysis of uis4(−) parasites. Anopheles stephensi mosquitoeswere raised under a 14 h light/10 h dark cycle at 28° C., 75% humidityand were fed on 10% sucrose solution. Blood-feeding and mosquitodissection was as described (35). The number of sporozoites per infectedmosquito was determined in a hemocytometer. To analyze sporozoitemotility, sporozoites were deposited onto precoated glass coverslips andincubated using primary antibody against P. berghei circumsporozoiteprotein (anti-PbCSP) (35). Bound antibody was detected using Alexa Fluor488-conjugated anti-mouse antibody (Molecular Probes). To detect liverstages in hepatocytes, P. berghei sporozoites were added to subconfluenthepatocytes, incubated 2 h at 37° C., and washed off. After 12, 24, 36and 48 h, liver stages were revealed using primary antibodies againstparasite heat shock protein 70 (HSP70) and a secondary antibodyconjugated with Alexa Fluor 488 (Molecular Probes). To analyzesporozoite invasion, 3×10⁴ salivary gland sporozoites were added tosubconfluent HepG2 cells and incubated for 90 min at 37° C. The ratiobetween intracellular and extracellular parasites was visualized using adouble staining protocol with the anti-CSP antibody (36) and confocalmicroscopy. To determine the infectivity of clonal sporozoitepopulations in vivo, C57/B16 mice were injected intravenously orsubcutaneously with 100sporozoite suspension of WT parasites or knockoutparasites in RPMI 1640. Parasitemia of the animals was checked daily byexamination of a Giemsa-stained blood smear. The appearance of a singleerythrocytic stage represents the first day of patency.

Immunization and parasite challenge experiments. For all experimentsfemale C57BL/6 mice (Charles River Laboratories) aged between 50 and 80days were used. For immunizations, uis4REP(−) sporozoites were extractedfrom the salivary glands from infected mosquitoes. Sporozoites wereinjected in a volume of 100 microliters intravenously into the tail veinof the animals. Animals were immunized with a single dose of 10,000 or50,000 uis4REP(−) sporozoites, followed by two boosts of either 10,000or 25,000 uis4REP(−) sporozoites adminstered i.v. The first boost wasgiven 14 days following the immunization, with a second boost following14 days thereafter. The animals were then monitored for parasitemia bydaily blood smears. Only those animals that remained blood stageparasite-negative after the first immunization and subsequent boostswere exposed to a challenge with WT sporozoites. Animals were challenged10 days after receiving the last boost of uis4REP(−) sporozoites byintravenous injection. All challenges consisted of 50,000 infective P.berghei WT sporozoites. For both sets of experiments, 5 naive animalswere included to verify infectivity of the sporozoite challenge dose. Ineach naive animal, parasitemia was readily detectable at day 3 afterinjection. Starting from day 3 after WT challenge, the uis4REP(−)sporozoite-immunized animals were examined for detectable parasitemia inGiemsa-stained blood smears. Animals did not show a detectableparasitemia within 50 days following the challenge and were consideredcompletely protected.

Results are shown in Table 2 below. Immunization with uis4REP(−)sporozoites confers sterile protection. The fact that a large proportionof mice remained blood stage negative after inoculation with uis4REP(−)sporozoites allowed us to test if immunization with these attenuatedsporozoites would protect mice against WT sporozoite challenge.Therefore, we immunized C57/b16 mice with 3 doses of 50,000 or 10,000uis4REP(−) sporozoites and subsequently challenged the mice, whichremained blood stage negative after immunization, with 50,000 infectiousWT sporozoites (Table 2). None of the immunized mice developed bloodstage infections after challenge and therefore enjoyed complete, sterileprotection. Naive mice that were challenged with 50,000 WT sporozoitesdeveloped blood stage infections 3 days after inoculation.

Table 2. C57B1/6 mice immunized with uis4REP(−) sporozoites arecompletely protected against a challenge with WT sporozoites.

TABLE 2 Immunization # Protected/ (uis4REP(—) Boosts # Challenged spz.)(days after immun./# of spz.) (prepatency) 50,000 1^(st) (14/25,000),2^(nd) (28/25,000) 8/8 (no infection)¹ none none 0/5 (day 3)² 10,0001^(st) (14/10,000), 2^(nd) (28/10,000) 8/8 (no infection)¹ none none 0/5(day 3)² Notes: ¹Immunized mice were challenged with 50,000 WT P.berghei sporozoites at day 38 after immunization. Mice were from thesame age group and sporozoites were from the same mosquito batch. Bloodsmears were evaluated up to day 50 after challenge. ²Naive control micewere from the same age group and challenged with 50,000 WT P. bergheisporozoites.

Our findings demonstrate that malaria parasites harbor genes that arenecessary only for successful completion of the pre-erythrocyticmammalian infection, within hepatocytes. We have shown that deletion ofsuch a gene effectively creates genetically attenuated malaria parasitesthat infect the liver of the mammalian host but are severely impaired intheir ability to further progress through the life cycle and causemalaria disease. Other genes in the Plasmodium genome, which arecritical for liver stage development, can be identified with thematerials and methods described herein.

Finally, we have shown here that immunization with UIS4 knockoutsporozoites confers complete, sterile protection against subsequentinfectious sporozoite challenge in a mouse model. This demonstrates thesuccessful use of genetically attenuated Plasmodium parasites as liveexperimental vaccines. Genetically attenuated human Plasmodium parasitesmay be similarly prepared as whole organism vaccines against malaria.

EXAMPLE 3

This third example describes a representative protocol for making aUIS3-like knockout in P. falciparum.

The P. falciparum UIS3 gene is targeted for disruption by replacementvia a well-known double-crossover recombination strategy. The UIS3 locusis replaced by a fragment containing the 5′ and 3′ untranslated regionsof the P. falciparum UIS3 open reading frame, each flanking the humandihydrofolate reductase (hdhfr) selectable marker. Sequence data for theP. falciparum UIS3 locus were obtained from the PlasmoDB database(www.plasmodb.org). The accession number for the coding sequence of P.falciparum UIS3 is PF13_(—)0012 (12) and the location of the exon withinchromosome 13 is 123930-124619 on the minus strand. The P. falciparumUIS3 rep1 fragment extends from nucleotides 124609-125594, and the rep2fragment from 122872-123921.

PfUIS3 rep 1 and 2 fragments are amplified from P. falciparum 3D7genomic DNA using Expand polymerase and the following primers: PfuIS3rep1 forward 5′-GAGTAATATAATGTGTAATGCATATGG-3′ (SEQ ID NO:15) andreverse 5′-GAGACCTTCATTTCAAAAAGGAAG-3′ (SEQ ID NO:16); PfUIS3 rep2forward 5′- CAAATGAAAACTTGGAAATAATCAGACGAG-3′ (SEQ ID NO:17) and reverse5′- GTATTATGCTTAAATTGGAAAAAAGTTTGAAG-3′ (SEQ ID NO:18). The sizes of therep1 and rep2 fragments amplified are 986 and 1051 base pairs,respectively. The PCR conditions are: one cycle of 94° C. for 3 min,followed by thirty cycles of 94° C. for 30 sec, 54.5° C. for 1 min, and65° C. for 3 min.

The PCR products are digested and cloned into the pHTK (47) vector. Rep1was cloned into restriction sites BgIII and SacII, and rep2 into EcoIand SfoI sites. The PfUIS3 replacement construct is sequenced to confirmcorrect cloning. Positive selection for transfected parasites carryingthe dhfr gene is carried out with the drug WR99210. pHTK contains thegene for thymidine kinase, allowing for negative selection of parasitescarrying the plasmid episomally.

A similar protocol may be used for making a knockout of any gene ofinterest in P. falciparum (for example, a UIS4-like gene, accessionnumber NP_(—)700638, PF10_(—)0164), or for making a knockout of suchgenes in other Plasmodium organisms. Genomic information, includinggenomic sequences, ESTs, annotations, automated predictions, SAGE tags,microarray data, mapping data, and open reading frames, for manyPlasmodium organisms, including, for example, P. falciparum, P. vivax,P. knowlesi, P. yoelii, P. chabaudi, P. reichenowi, and P. gallinaceum,is readily available in public databases such as the National Center forBiotechnology Information (www.ncbi.nlm.nih.gov), the Plasmodium GenomeDatabase (www.plasmodb.org), and the Sanger Institute(www.sanger.ac.uk).

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While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1. A live Plasmodium organism that is genetically engineered to disrupta gene whose expression is up-regulated in infective salivary glandsporozoites and whose function is not required for entry into hosthepatocytes.
 2. A live Plasmodium organism that is geneticallyengineered to disrupt a gene whose expression is up-regulated inliver-stage parasites and whose function is not required for entry intohost hepatocytes.